This Program combines two powerful analytical approaches to discover the causes of spontaneous mutations in human cells, yeasts and bacteria. The first approach involves a major technical advance in mutational spectrometry which permits measurement of predominate point mutations without reference to changes in phenotype. The second approach is the use of biochemical genetics aimed with the many known mutant genes for DNA replication and DNA repair in yeasts and bacteria and the growing set of such genes known in humans. By using such mutants and observing changes in spontaneous mutational spectra we expect to gain important new mechanistic understanding. A series of specific hypotheses regarding the role of DNA manipulation or endogenous DNA damage are presented throughout five complementary projects. The Program's direction is strongly influenced by a growing body of evidence that mutations in human germinal and somatic cells contain a significant fraction of mutations which appear to be spontaneous in origin. The Program's five projects share: -a common focus on spontaneous mutation mechanisms, -a set of common DNA sequences found in the human genome and genetically engineered into yeasts and bacteria -a common technological tool-constant denaturant gradient electrophoresis/high fidelity PCR -applied to obtain point mutational spectra.